依维莫司协同全反式维甲酸对耐药急性早幼粒细胞白血病的作用及机制研究
发布时间：2019-03-23 17:45
【摘要】：目的：急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)是髓系细胞停止在早幼粒细胞阶段的一类较为特殊的急性髓系白血病。随着全反式维甲酸(all-trans retinoid acid,ATRA)常规用于APL的治疗后,APL患者的预后得到了显著提高。但是,单用ATRA后耐药问题也日益严重。依维莫司(Everolimus, RAD001)一种新型哺乳动物雷帕霉素受体(mTOR)抑制剂,为雷帕霉素的衍生物,具有免疫抑制、抗肿瘤、抗病毒等多种作用。因此,本研究旨在探讨依维莫司协同ATRA对APL的作用及机制。方法：用RAD001和ATRA分别或联合处理急性早幼粒细胞白血病细胞株NB4-R1细胞。应用CDllb染色流式细胞术及硝基四唑氮蓝(NBT)还原实验检测细胞分化,运用CCK-8法检测细胞增殖情况,周期试剂盒流式检测细胞周期情况,AnnexinV/PI双染色检测细胞凋亡情况,免疫印迹法检测自噬相关蛋白LC3、Beclinl,凋亡相关蛋白caspase家族,周期相关蛋白CyclinD1、P27Kip1及其他P70S6K、 P-P70S6K、4E-BP1、P-4E-BP1、PML-RARa等蛋白质表达水平,RT-PCR方法检测分化相关基因C/EBPε的mRNA表达水平。结果：(1)用1nM、10nM、100nM的RAD001和1μM的ATRA分别或联合处理NB4-R1细胞24h、48h、72h后。流式细胞术和NBT还原实验显示,联合用药组能够明显诱导NB4-R1细胞的分化,而单独使用RAD001或ATRA并不能有效诱导NB4-R1细胞的分化。其中100nM的RAD001联合1μM的ATRA和单独1μM的ATRA处理NB4-R1细胞48h后分化比率分别为(54.47±4.91)%和(17.07±2.65)%(p0.01)。(2)用1nM、10nM、100nM的RAD001和1 μM的ATRA分别或联合处理NB4-R1细胞48h后。CCK-8法检测药物对细胞的增殖情况发现,联合用药明显抑制细胞的增殖(p0.01),并且随着联用RAD001的浓度的增加,抑制作用也逐渐增强。而单独用药对细胞的抑制作用不明显。(3)用浓度为100nM的RAD001和1μM的ATRA分别或联合处理APL细胞株NB4-R1细胞48h后,流式细胞术周期检测联合用药组G1期细胞明显增多,而S期细胞明显减少(p0.05)。免疫印迹法结果显示联合用药后细胞周期相关蛋白cyclinD1表达相对单独用ATRA组明显下调,而P27Kip1蛋白表达水平上调。(4)用浓度为100nM的RAD001和1μM的ATRA分别或联合处理APL细胞株NB4-R1细胞48h后,流式细胞术凋亡检测发现联合用药组和单独用药组细胞并未发生明显凋亡。免疫印迹法检测凋亡相关蛋白Caspase家族caspase3、caspase8、 caspase9发现各组之间并未发生明显变化。(5)用浓度为100nM的RAD001和1μM的ATRA分别或联合处理APL细胞株NB4-R1细胞48h后,RT-PCR检测分化相关基因C/EBPε的mRNA水平显示联合用药组相对与单独用药组明显上调(p0.01)。免疫印迹检测PML-RARa融合蛋白发现,联合用药使融合蛋白表达下降, RAD001协同ATRA明显抑制了mTOR下游底物P70S6K、E-BP1的磷酸化,自噬相关蛋白LC3-Ⅱ、Beclinl表达明显上调。而加入10mM的自噬抑制剂3-MA处理后,APL细胞株NB4-R1细胞分化水平明显下降(p0.01)并且可逆转联合用药对PML-RARa融合蛋白的部分降解作用。结论：(1)RAD001协同ATRA可以诱导耐药的急性早幼粒细胞白血病细胞株NB4-R1细胞分化,达到逆转其耐药的作用。可能是通过上调转录因子C/EBPε的表达和抑制mTOR信号通路诱导NB4-R1发生自噬部分降解PML-RARa融合蛋白等多种途径来实现的。(2)RAD001协同ATRA明显抑制NB4-R1细胞的增殖。(3)RAD001协同ATRA使NB4-R1细胞的细胞周期被阻断在G1期,S期细胞减少,可能与联合用药下调CyclinD1蛋白,上调P27Kip1蛋白有关。(4)RAD001协同ATRA作用NB4-R1细胞后,细胞不发生明显的凋亡,对凋亡相关蛋白Caspase家族没有影响,因此联合用药不影响细胞的生存。
[Abstract]:Objective: Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia in the stage of early promyelocytic leukemia. With all-trans retinoic acid (ATRA) in the treatment of APL, the prognosis of APL patients has been improved significantly. However, the problem of drug resistance after ATRA alone is also increasing. Everolimus (RAD001), a novel mammalian rapamycin receptor (mTOR) inhibitor, is a derivative of rapamycin, and has various effects such as immunosuppression, anti-tumor, and anti-virus. Therefore, the purpose of this study is to explore the effect and mechanism of everolimus-based ATRA on APL. Methods: The cells of acute promyelocytic leukemia cell line NB4-R1 were treated with RAD001 and ATRA, respectively. The cell differentiation was detected by means of CD11b staining flow cytometry and NBT reduction. The cell proliferation was detected by CCK-8 method. The cell cycle and Annexin V/ PI double staining were used to detect the cell cycle, and the autophagy-related proteins LC3 and Beclinl were detected by immunoblotting. The expression levels of apoptosis-related protein caspase-family, cyclin D1, P27Kip1 and other P70S6K, P-P70S6K, 4E-BP1, P-4E-BP1, PML-RARa were detected by RT-PCR. Results: (1) NB4-R1 cells were treated with 1 nM,10 nM,100 nM RAD001 and 1. m u.M ATRA respectively or in combination with NB4-R1 cells for 24 h,48 h, and 72 h. Flow cytometry and NBT reduction experiments show that the combination group can obviously induce the differentiation of NB4-R1 cells, and the use of RAD001 or ATRA alone can not effectively induce the differentiation of NB4-R1 cells. The rate of differentiation was 54.47 (4.91)% and (17.07% 2.65)% (p0.01) in 100 nM of RAD001 in combination with 1.mu. M of ATRA and 1. m (2) NB4-R1 cells were treated with 1 nM,10 nM,100 nM of RAD001 and 1.mu. M of ATRA, respectively, or in combination with NB4-R1 cells for 48 h. The proliferation of the cells was detected by the CCK-8 method, and the combined administration of the drug significantly inhibited the proliferation of the cells (p0.01), and the inhibition was also enhanced with the increase of the concentration of the combination of RAD001. And the inhibition effect of the individual medicine on the cells is not obvious. (3) After 48 h of the APL cell line NB4-R1 cells were treated with ATRA with a concentration of 100 nM and ATRA of 1. m u.M, respectively, the number of G1 cells in the combined administration group was significantly increased by the flow cytometry cycle, and the S-phase cells were significantly reduced (p0.05). The results of immunoblotting showed that the expression of cyclinD1 in the cell cycle after combined administration was significantly reduced with the ATRA group, while the level of P27Kip1 protein was up-regulated. (4) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the cell apoptosis was detected by flow cytometry. The expression of caspase-3, caspase-8 and caspase-9 in the apoptosis-related protein was detected by immunoblotting. (5) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the mRNA level of the related gene C/ EBP was significantly up-regulated by RT-PCR (p0.01). The fusion protein of PML-RARa was detected by immunoblotting. In combination, the expression of fusion protein was decreased, and the co-administration of RAD001 and ATRA significantly inhibited the phosphorylation of P70S6K, E-BP1 in the downstream substrate of mTOR, and the expression of autophagy-related protein LC3-鈪，
本文编号：2446092
[Abstract]:Objective: Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia in the stage of early promyelocytic leukemia. With all-trans retinoic acid (ATRA) in the treatment of APL, the prognosis of APL patients has been improved significantly. However, the problem of drug resistance after ATRA alone is also increasing. Everolimus (RAD001), a novel mammalian rapamycin receptor (mTOR) inhibitor, is a derivative of rapamycin, and has various effects such as immunosuppression, anti-tumor, and anti-virus. Therefore, the purpose of this study is to explore the effect and mechanism of everolimus-based ATRA on APL. Methods: The cells of acute promyelocytic leukemia cell line NB4-R1 were treated with RAD001 and ATRA, respectively. The cell differentiation was detected by means of CD11b staining flow cytometry and NBT reduction. The cell proliferation was detected by CCK-8 method. The cell cycle and Annexin V/ PI double staining were used to detect the cell cycle, and the autophagy-related proteins LC3 and Beclinl were detected by immunoblotting. The expression levels of apoptosis-related protein caspase-family, cyclin D1, P27Kip1 and other P70S6K, P-P70S6K, 4E-BP1, P-4E-BP1, PML-RARa were detected by RT-PCR. Results: (1) NB4-R1 cells were treated with 1 nM,10 nM,100 nM RAD001 and 1. m u.M ATRA respectively or in combination with NB4-R1 cells for 24 h,48 h, and 72 h. Flow cytometry and NBT reduction experiments show that the combination group can obviously induce the differentiation of NB4-R1 cells, and the use of RAD001 or ATRA alone can not effectively induce the differentiation of NB4-R1 cells. The rate of differentiation was 54.47 (4.91)% and (17.07% 2.65)% (p0.01) in 100 nM of RAD001 in combination with 1.mu. M of ATRA and 1. m (2) NB4-R1 cells were treated with 1 nM,10 nM,100 nM of RAD001 and 1.mu. M of ATRA, respectively, or in combination with NB4-R1 cells for 48 h. The proliferation of the cells was detected by the CCK-8 method, and the combined administration of the drug significantly inhibited the proliferation of the cells (p0.01), and the inhibition was also enhanced with the increase of the concentration of the combination of RAD001. And the inhibition effect of the individual medicine on the cells is not obvious. (3) After 48 h of the APL cell line NB4-R1 cells were treated with ATRA with a concentration of 100 nM and ATRA of 1. m u.M, respectively, the number of G1 cells in the combined administration group was significantly increased by the flow cytometry cycle, and the S-phase cells were significantly reduced (p0.05). The results of immunoblotting showed that the expression of cyclinD1 in the cell cycle after combined administration was significantly reduced with the ATRA group, while the level of P27Kip1 protein was up-regulated. (4) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the cell apoptosis was detected by flow cytometry. The expression of caspase-3, caspase-8 and caspase-9 in the apoptosis-related protein was detected by immunoblotting. (5) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the mRNA level of the related gene C/ EBP was significantly up-regulated by RT-PCR (p0.01). The fusion protein of PML-RARa was detected by immunoblotting. In combination, the expression of fusion protein was decreased, and the co-administration of RAD001 and ATRA significantly inhibited the phosphorylation of P70S6K, E-BP1 in the downstream substrate of mTOR, and the expression of autophagy-related protein LC3-鈪，
本文编号：2446092
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